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A mixture of seaweed extracts and glycosaminoglycans from sea squirts inhibits α-MSH-induced melanogenesis in B16F10 melanoma cells

A mixture of seaweed extracts and glycosaminoglycans from sea squirts inhibits α-MSH-induced... Background: In the present study, the skin-whitening effects of a marine-sourced mixture that includes a fucoidan- rich extract of Undaria pinnatifida (UPEF), a phlorotannin-rich extract of Ecklonia cava (ECE), and glycosaminoglycans (GAGs) from sea squirt skin were investigated. Methods: The whitening effects of the mixture and its components were evaluated by measuring the inhibition of mushroom tyrosinase and melanin synthesis in alpha-melanocyte-stimulating hormone (α-MSH)-stimulated B16F10 melanoma cells. Results: Each component alone markedly inhibited mushroom tyrosinase in a dose-dependent manner, and in α- MSH-stimulated B16F10 cells, they inhibited melanin synthesis and were cytotoxic. However, the whitening effects of UPEF, ECE, and GAGs in combination were greater than those of each component alone. A mixture in the ratio of 4:5:1 (UEG-451) showed the strongest activity without cytotoxicity. Further study suggested that UEG-451 inhibits α-MSH-stimulated melanogenesis in B16F10 cells by downregulating tyrosinase and tyrosinase-related proteins, such as TRP-1 and TRP-2, via the inhibition of MITF expression. Conclusions: These results suggest that mixing the different components at optimum ratios might be an effective way to improve their bioactivities and reduce toxicity and that UEG-451 possesses strong whitening effects that could be used in the cosmetic industry. Keywords: Marine-sourced mixture, Seaweed extracts, Melanogenesis, Whitening Background has no side effects, may be a potential candidate from Melanogenesis is the physiological process that produces which to develop a therapeutic agent or cosmetic. melanin pigment, which contributes to skin and hair The ocean is an abundant source of both biologically color (Gilchrest and Eller 1999; Kim et al. 2013). Mel- and chemically diverse species. Because of the special anin is the key pigment responsible for skin color in environment, marine organisms, including plants, ani- humans. Melanin may be overproduced in melasma, mals, and microorganisms, produce unique metabolites ultraviolet irradiation, and hyperpigmentation diseases. (Kijjoa and Sawangwong 2004; Wang et al. 2016). These Recently, an increasing number of women desire whiter metabolites, such as phenolic compounds, carbohydrates, complexions, especially in Asian countries (Tengamnuay and peptides, possess antioxidant, anti-inflammatory, anti- et al. 2006). Therefore, a naturally sourced compound, cancer, anti-obesity, anti-hypertensive, and anti-diabetes which not only downregulates melanogenesis but also bioactivities (Fernando et al. 2017;Kang et al. 2015;Kang et al. 2013; Kim et al. 2016; Kim et al. 2014;Koet al. 2017; Lange et al. 2015; Lee et al. 2015; Lee et al. 2013;Ohetal. * Correspondence: herolegend@hanmail.net; youjinj@jejunu.ac.kr 2016;Samarakoonet al. 2014; Sanjeewa et al. 2016). Ahn Lei Wang and Yong Ri Cui contributed equally to this work. Department of Marine Life Sciences, Jeju National University, Jeju 63243, et al. (2007) reported on the antioxidant activities of Republic of Korea © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wang et al. Fisheries and Aquatic Sciences (2019) 22:11 Page 2 of 8 phlorotannins purified from Ecklonia cava, an edible purified; the purity of GAGs was 95%. All samples brown alga (Ahn et al. 2007). Ko et al. (2016)purified pep- were stored at − 20 °C until use. The sample mixtures tides from flounder and investigated the anti-hypertensive were prepared by mixing each component solution at activities of those peptides (Ko et al. 2016). Kim et al. the described ratios. (2014) isolated active compounds from marine bacteria and evaluated their bioactivities (Kim et al. 2014). Undaria pinnatifida, an edible brown alga, is rich in Measurement of the inhibitory effects of UPEF, ECE, and polysaccharides, especially fucoidan. Park and Choi GAGs and their mixtures on mushroom tyrosinase (2017) isolated different molecular weight fucoidan The inhibition of mushroom tyrosinase was measured as from U. pinnatifida and investigated their radical previously described (Heo et al. 2009; Kang et al. 2012). scavenging activities and inhibition of melanogenesis In brief, the 200-μL assay mixture in a 96-well micro- (Park and Choi 2017). E. cava is rich in phlorotan- plate contained 40 μL of 1.5-mM L-tyrosine, 140 μLof nins, and we reported their antioxidant and melano- 50-mM phosphate buffer (pH 6.5), 10 μL of aqueous genesis inhibitory activities in a previous study (Ahn mushroom tyrosinase (1000 units/mL), and 10 μL of test et al. 2007; Heo et al. 2009). Glycosaminoglycans solution. The assay mixture was incubated at 37 °C for (GAGs) are long, unbranched sulfated polysaccha- 12 min and then kept on ice for 5 min to stop the rides that possess strong antioxidant activity and reaction. The amount of dopachrome in the reaction possess the potential in cosmetic area (Campo et al. mixture was measured at 490 nm using a microplate 2004). The objectives of the present study were to reader (BioTek Synergy HT, BioTek Instruments, evaluate the whitening effects of a fucoidan-rich ex- Winooski, VT, USA). tract from U. pinnatifida, a phlorotannin-rich extract from E. cava, and GAGs from sea squirt skin. To Cell culture improve the whitening effects and to reduce the cy- B16F10 mouse melanoma cells (ATCC® CRL-6475™) totoxicities of these compounds, they were mixed in were purchased from ATCC (American Type Culture different ratios and tested to select the mixture that Collection, Manassas, VA, USA) and grown in conferred optimal whitening without cytotoxicity. DMEM supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were incubated in an atmosphere of 5% Materials and methods CO at 37 °C and were sub-cultured every 4 days. Chemicals and reagents Cells for experiments were seeded at a concentration Dimethyl sulfoxide (DMSO), 3-(4-5-dimethyl-2yl)-2-5- of 5 × 10 cells/mL. diphynyltetrasolium bromide (MTT), mushroom tyrosin- ase, and alpha-melanocyte-stimulating hormone (α-MSH) were purchased from Sigma Co. (St. Louis, MO, USA). Cell viability assay Dulbecco’s modified Eagle medium (DMEM), penicillin/ Cell viability was quantified by a colorimetric MTT streptomycin, and fetal bovine serum (FBS) were pur- assay (Wang et al. 2017;Wangetal. 2018). Briefly, chased from Gibco BRL (Life Technologies, Burlington, B16F10 cells were seeded in a 96-well plate and incu- ON, Canada). Antibodies against tyrosinase, tyrosinase- bated for 24 h. The cells were treated with different related protein-1 and protein-2 (TRP-1 and TRP-2), and concentrations of test samples and incubated for 72 h. microphthalmia-associated transcription factor (MITF) MTT solution (50 μL, 2 mg/mL) was added to each were purchased from Santa Cruz Biotechnology (Santa well and incubated for 3 h. The supernatant was aspi- Cruz, CA, USA). Anti-mouse and anti-rabbit IgG were rated, 150 μL of DMSO was added to each well, and purchased from Cell Signaling Technology (Beverly, MA, the absorbance was measured at 540 nm using a mi- USA). All other chemicals were of analytical grade. croplate reader. Preparation of fucoidan-rich extract, phlorotanin-rich extract, and glycosaminoglycans Measurement of cellular melanin content U. pinnatifida was hydrolyzed with Celluclast. The B16F10 cells were seeded in a 6-well plate and incu- polysaccharide fraction was precipitated with ethanol bated for 24 h. The cells were treated with various con- and referred to as UPEF. UPEF contains 36.10 ± 3.20% centrations of test samples and stimulated with α-MSH fucoidan. An 80% ethanol extract of E. cava was pre- (50 nM). After 72 h, the cells were washed with ice-cold pared (ECE), and it contained 26.85 ± 0.16% phenols. PBS and harvested. The harvested cells were incubated The sea squirt skin was hydrolyzed with Celluclast, at 80 °C for 1 h in 1 mL of 1-N NaOH containing 10% and glycosaminoglycans (GAGs) were separated and DMSO. The absorbance of the supernatant was Wang et al. Fisheries and Aquatic Sciences (2019) 22:11 Page 3 of 8 measured at 490 nm using a microplate reader (Heo et Results al. 2010). Tyrosinase inhibition by UPEF, ECE, and GAGs The effects of UPEF, ECE, and GAGs on tyrosinase activity were examined by measuring L-tyrosine hydroxyl- Western blot analysis ation. Arbutin was used as a positive control. As Fig. 1 The effect of the test samples on the expression of shows, UPEF, ECE, and GAGs inhibited tyrosinase activity melanogenesis-related proteins including MITF, tyrosinase, in a dose-dependent manner. ECE showed the strongest TRP-1, and TRP-2 was assessed by Western blot analysis as inhibition of the three agents, and it inhibited tyrosinase described previously (Kim et al. 2013). In brief, B16F10 cells activity by 64.33% at a concentration of 100 μg/mL. were incubated with various concentrations of the test sam- ple and stimulated with α-MSH (50 nM). After 72 h, cells were harvested and lysed. The protein content of each sam- Cytotoxicity of UPEF, ECE, and GAGs on B16F10 cells ple was measured with a BCA™ kit. The proteins (50 μg) The cytotoxicities of UPEF, ECE, and GAGs on B16F10 were separated by SDS-PAGE and transferred onto nitro- cells were examined by MTT assay, and the results are cellulose membranes. The membranes were incubated in summarized in Fig. 2a. As the results show, ECE caused blocking buffer (5% skim milk) and then with primary anti- significant cytotoxicity in B16F10 cells. In addition, bodies for 16 h at 4 °C. The membranes were then incu- UPEF and GAGs were slightly toxic to B16F10 cells at bated with secondary antibody at room temperature for 3 high concentration (100 μg/mL). h. Finally, the proteins were visualized using an ECL West- ern blotting detection kit and exposure to X-ray film. Effects of UPEF, ECE, and GAGs on melanin synthesis in α- MSH-stimulated B16F10 cells Statistical analysis As Fig. 2b indicates, the melanin content of cells not All experiments were performed in triplicate. The data were stimulated with α-MSH is referred to as 100%, and expressed as means ± standard errors (S.E). The mean the melanin content of cells stimulated with α-MSH values of each experiment werecompared using one-way increased by 80%. The melanin content of cells ANOVA. Significant differences between the means were treated with UPEF, ECE, and GAGs decreased in a determined by the Duncan test. A p value < 0.05 was dose-dependent manner. These results indicated that considered to be statistically significant, and degrees of sig- all three test substances inhibited melanogenesis in α- nificance were indicated as follows: *p < 0.05, **p <0.01, MSH-stimulated B16F10 cells, and ECE showed the ### ***p < 0.001, and p <0.001. strongest effect. Fig. 1 Effect of UPEF, ECE, and GAGs on mushroom tyrosinase activity. Tyrosinase activity was measured by colorimetric assay. Dopachrome was measured at 490 nm using a microplate reader. The data are expressed as the means ± S. E (n =3) Wang et al. Fisheries and Aquatic Sciences (2019) 22:11 Page 4 of 8 Fig. 2 Cytotoxicity and melanin synthesis in UPEF-, ECE-, and GAGs-treated B16F10 cells. a Cytotoxicity on B16F10 cells; b melanin synthesis in α- MSH-stimulated B16F10 cells. The data are expressed as the means ± S. E (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 as compared to the α- ### MSH-treated group and p < 0.001 as compared to the control group Tyrosinase inhibition by combinations of UPEF, ECE, and combinations in B16F10 cells were measured by MTT GAGs assay. As Fig. 4a shows, most combinations were UPEF, ECE, and GAGs were combined in various ratios slightly toxic to B16F10 cells, and UPEF to ECE to by volume, and the effect on tyrosinase activity was mea- GAGs at 2:6:2 (UEG-262) showed the strongest cyto- sured. As Fig. 3 shows, tyrosinase activity decreased as toxicity. On the other hand, UEG-451 showed no cyto- the ECE ratio in the mixture increased. In addition, the toxicity in B16F10 cells. inhibitory activities of the test agents in combination were stronger compared to each agent alone at the same concentration. Effect of test agents in combination on melanin synthesis in α-MSH-stimulated B16F10 cells The effects of the test agents in combination on melano- Cytotoxicity of UPEF, ECE, and GAGs in combination on genesis were assessed by measuring melanin synthesis in B16F10 cells α-MSH-stimulated B16F10 cells. As the results show Using the tyrosinase inhibition conferred by the test (Fig. 4b), all combinations significantly reduced melanin agents in combination, seven combinations (UEG-262, synthesis in α-MSH-stimulated B16F10 cells, especially UEG-271, UEG-352, UEG-361, UEG-433, UEG-451, UEG-451. These results show that UPEF, ECE, and and UEG-721) were selected to test the effects on mela- GAGs in combination inhibited melanogenesis, and 4:5: nogenesis in B16F10 cells. The cytotoxicities of these 1 is the optimum ratio. Wang et al. Fisheries and Aquatic Sciences (2019) 22:11 Page 5 of 8 Fig. 3 The effects of UPEF, ECE, and GAGs in combination on mushroom tyrosinase activity. Tyrosinase activity was measured by colorimetric assay. Dopachrome was measured at 490 nm using a microplate reader. The data are expressed as the means ± S. E (n =3) Fig. 4 Cytotoxicity and melanin synthesis in B16F10 cells treated with UPEF, ECE, and GAGs in combination. a Cytotoxicity on B16F10 cells; (b) melanin synthesis in α-MSH-stimulated B16F10 cells. The data are expressed as the means ± S. E (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 as ### compared to the α-MSH-treated group and p < 0.001 as compared to the control group Wang et al. Fisheries and Aquatic Sciences (2019) 22:11 Page 6 of 8 Fig. 5 Effect of UEG-451 on tyrosinase, TRP-1, TRP-2, and MITF expression in α-MSH-stimulated B16F10 cells. a The effect of UEG-451 on tyrosinase, TRP-1, TRP-2, and MITF expressions; (b) the relative amounts of tyrosinase, TRP-1, TRP-2, and MITF. The relative amounts of tyrosinase, TRP-1, TRP-2, and MITF were normalized to β-actin. The data are expressed as the means ± S. E (n = 3). **p < 0.01 and ***p < 0.001 as compared to ### the α-MSH-treated group and p < 0.001 as compared to the control group Effect of UEG-451 on tyrosinase, TRP-1, TRP-2, and MITF are made from natural or chemosynthetic materials, are expression in α-MSH-stimulated B16F10 cells commercially available. However, some of these mate- Based on its cytotoxicity and inhibition of melanogenesis, rials possess side effects or are toxic because they con- UEG-451 was selected for further research to evaluate the tain substances such as hydroquinone and heavy metals. mechanisms of its anti-tyrosinase and anti-melanogenesis Thus, more research is devoted to searching for safe and activities. The effect of UEG-451 on the expression of effective whitening agents from natural sources. tyrosinase, TRP-1, TRP-2, and MITF was examined by Many studies have reported the effects of plant ex- Western blot analysis. As shown in Fig. 5, the expressions tracts or compounds isolated from plants on melanogen- of tyrosinase, TRP-1, TRP-2, and MITF were increased by esis (Arung et al. 2011; Chan et al. 2011). Arung et al. α-MSH stimulation and the expressions of these proteins (2011) isolated quercetin and its derivatives from Allium were reduced in cells pre-treated with different concentra- cepa and investigated their anti-melanogenesis effects tions of UEG-451. (Arung et al. 2011). Chan et al. (2011) evaluated the tyrosinase inhibition and melanin synthesis inhibition in Discussion B16F10 cells of Sargassum polycystum ethanolic extracts In Asian cultures, lighter skin tones are considered more and their fractions (Chan et al. 2011). In the present desirable. To meet the needs of the many women who study, we evaluated melanogenesis inhibition conferred by suffer from hyperpigmentation, whitening agents have a fucoidan-rich extract, a phlorotannin-rich extract, and been developed for cosmetic and medical applications. GAGs obtained from seaweed and sea squirt, and investi- Various whitening cosmetics or cosmeceuticals, which gated their whitening effects when in combination. Wang et al. Fisheries and Aquatic Sciences (2019) 22:11 Page 7 of 8 The effects of UPEF, ECE, and GAGs on commercial three agents inhibited tyrosinase and melanin synthesis mushroom tyrosinase were investigated, and the results in α-MSH-stimulated B16F10 cells and that they can act indicated that all samples possess tyrosinase inhibitory ac- synergistically when in combination. In addition, UPEF, tivity with ECE showing the strongest activity of the three ECE, and GAGs in the ratio of 4:5:1 (UEG-451) showed (Fig. 1). In addition, UPEF, ECE, and GAGs significantly the strongest activity and was not toxic. These results reduced α-MSH-induced melanin synthesis in B16F10 suggest UEG-451 may be an ideal whitening agent for cells in a dose-dependent manner (Fig. 2). ECE showed use in the medical and cosmetic industries. stronger melanin synthesis inhibition activity than UPEF Acknowledgements and GAGs, and the melanin content of cells treated with The authors wish to thank the Ministry of Trade, Industry and Energy 100 μg/mL of ECE was lower than the unstimulated cells. (MOTIE), and Korea Institute for Advancement of Technology (KIAT) for the support for this study. These results demonstrated that ECE is a potent in- hibitor of melanin synthesis in α-MSH-stimulated and Authors’ contributions non-stimulated B16F10 cells. However, all three sam- LW, JYK, and YJJ designed this study and wrote the manuscript. LW, YRC, ples were cytotoxic to B16F10 cells, especially ECE. HWY, and HGL performed the experiments and analyzed the data. LW and YRC contributed equally to this study. All authors read and approved the Therefore, we mixed the test agents in different ratios final manuscript. and compared the whitening effects and toxicities of the combinations to those of the agents alone. Funding The inhibition of tyrosinase and melanin synthesis This research is supported by “Rediscover of the Past R&D Result” through the Ministry of Trade, Industry and Energy (MOTIE), and Korea Institute for conferred by the combinations was stronger than each Advancement of Technology (KIAT) (Grant No.: N0002151, Project: The single agent at the same concentrations (Fig. 1 and Fig. 3; commercialization of anti-aging products (Aquato Return 20). Fig. 2b and Fig. 4b); UEG-451 showed the strongest ac- Availability of data and materials tivity and no toxicity, with 71.10% of control tyrosinase All data sets generated and analyzed during the current study are available activity and a 115.24% reduction in α-MSH-stimulated from the corresponding author on reasonable request. melanin synthesis at 50 μg/mL. Combining the agents at an optimal ratio may be an ideal and effective way to im- Ethics approval and consent to participate Not applicable. prove bioactivity and reduce cytotoxicity. Melanogenesis is regulated by enzymes including tyro- Consent for publication sinase, TRP-1, and TRP-2. Tyrosinase is considered to Not applicable. be the rate-limiting enzyme of melanin biosynthesis and Competing interests represents the major regulatory step in melanogenesis The authors declare that they have no competing interests. (Maeda et al. 1997). Thus, the inhibition of related en- zymes is the most common approach to developing Received: 9 April 2019 Accepted: 17 May 2019 skin-whitening agents. The present results revealed that UEG-451 inhibited tyrosinase and reduced melanin syn- References thesis in α-MSH-stimulated B16F10 cells. In addition, Ahn G-N, Kim K-N, Cha S-H, Song C-B, Lee J, Heo M-S, Yeo I-K, Lee N-H, Jee Y-H, Western blot results demonstrated that the expressions Kim J-S. Antioxidant activities of phlorotannins purified from Ecklonia cava on free radical scavenging using ESR and H2O2-mediated DNA damage. Eur of tyrosinase, TRP-1, and TRP-2 in UEG-451-treated Food Res Technol. 2007;226:71–9. cells decreased in a dose-dependent manner compared Arung ET, Furuta S, Ishikawa H, Kusuma IW, Shimizu K, Kondo R. Anti- to non-treated cells (Fig. 5a and b). The tyrosinase fam- melanogenesis properties of quercetin-and its derivative-rich extract from Allium cepa. 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A mixture of seaweed extracts and glycosaminoglycans from sea squirts inhibits α-MSH-induced melanogenesis in B16F10 melanoma cells

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Springer Journals
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Copyright © 2019 by The Author(s)
Subject
Life Sciences; Fish & Wildlife Biology & Management; Marine & Freshwater Sciences; Zoology; Animal Ecology
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2234-1757
DOI
10.1186/s41240-019-0126-3
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Abstract

Background: In the present study, the skin-whitening effects of a marine-sourced mixture that includes a fucoidan- rich extract of Undaria pinnatifida (UPEF), a phlorotannin-rich extract of Ecklonia cava (ECE), and glycosaminoglycans (GAGs) from sea squirt skin were investigated. Methods: The whitening effects of the mixture and its components were evaluated by measuring the inhibition of mushroom tyrosinase and melanin synthesis in alpha-melanocyte-stimulating hormone (α-MSH)-stimulated B16F10 melanoma cells. Results: Each component alone markedly inhibited mushroom tyrosinase in a dose-dependent manner, and in α- MSH-stimulated B16F10 cells, they inhibited melanin synthesis and were cytotoxic. However, the whitening effects of UPEF, ECE, and GAGs in combination were greater than those of each component alone. A mixture in the ratio of 4:5:1 (UEG-451) showed the strongest activity without cytotoxicity. Further study suggested that UEG-451 inhibits α-MSH-stimulated melanogenesis in B16F10 cells by downregulating tyrosinase and tyrosinase-related proteins, such as TRP-1 and TRP-2, via the inhibition of MITF expression. Conclusions: These results suggest that mixing the different components at optimum ratios might be an effective way to improve their bioactivities and reduce toxicity and that UEG-451 possesses strong whitening effects that could be used in the cosmetic industry. Keywords: Marine-sourced mixture, Seaweed extracts, Melanogenesis, Whitening Background has no side effects, may be a potential candidate from Melanogenesis is the physiological process that produces which to develop a therapeutic agent or cosmetic. melanin pigment, which contributes to skin and hair The ocean is an abundant source of both biologically color (Gilchrest and Eller 1999; Kim et al. 2013). Mel- and chemically diverse species. Because of the special anin is the key pigment responsible for skin color in environment, marine organisms, including plants, ani- humans. Melanin may be overproduced in melasma, mals, and microorganisms, produce unique metabolites ultraviolet irradiation, and hyperpigmentation diseases. (Kijjoa and Sawangwong 2004; Wang et al. 2016). These Recently, an increasing number of women desire whiter metabolites, such as phenolic compounds, carbohydrates, complexions, especially in Asian countries (Tengamnuay and peptides, possess antioxidant, anti-inflammatory, anti- et al. 2006). Therefore, a naturally sourced compound, cancer, anti-obesity, anti-hypertensive, and anti-diabetes which not only downregulates melanogenesis but also bioactivities (Fernando et al. 2017;Kang et al. 2015;Kang et al. 2013; Kim et al. 2016; Kim et al. 2014;Koet al. 2017; Lange et al. 2015; Lee et al. 2015; Lee et al. 2013;Ohetal. * Correspondence: herolegend@hanmail.net; youjinj@jejunu.ac.kr 2016;Samarakoonet al. 2014; Sanjeewa et al. 2016). Ahn Lei Wang and Yong Ri Cui contributed equally to this work. Department of Marine Life Sciences, Jeju National University, Jeju 63243, et al. (2007) reported on the antioxidant activities of Republic of Korea © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wang et al. Fisheries and Aquatic Sciences (2019) 22:11 Page 2 of 8 phlorotannins purified from Ecklonia cava, an edible purified; the purity of GAGs was 95%. All samples brown alga (Ahn et al. 2007). Ko et al. (2016)purified pep- were stored at − 20 °C until use. The sample mixtures tides from flounder and investigated the anti-hypertensive were prepared by mixing each component solution at activities of those peptides (Ko et al. 2016). Kim et al. the described ratios. (2014) isolated active compounds from marine bacteria and evaluated their bioactivities (Kim et al. 2014). Undaria pinnatifida, an edible brown alga, is rich in Measurement of the inhibitory effects of UPEF, ECE, and polysaccharides, especially fucoidan. Park and Choi GAGs and their mixtures on mushroom tyrosinase (2017) isolated different molecular weight fucoidan The inhibition of mushroom tyrosinase was measured as from U. pinnatifida and investigated their radical previously described (Heo et al. 2009; Kang et al. 2012). scavenging activities and inhibition of melanogenesis In brief, the 200-μL assay mixture in a 96-well micro- (Park and Choi 2017). E. cava is rich in phlorotan- plate contained 40 μL of 1.5-mM L-tyrosine, 140 μLof nins, and we reported their antioxidant and melano- 50-mM phosphate buffer (pH 6.5), 10 μL of aqueous genesis inhibitory activities in a previous study (Ahn mushroom tyrosinase (1000 units/mL), and 10 μL of test et al. 2007; Heo et al. 2009). Glycosaminoglycans solution. The assay mixture was incubated at 37 °C for (GAGs) are long, unbranched sulfated polysaccha- 12 min and then kept on ice for 5 min to stop the rides that possess strong antioxidant activity and reaction. The amount of dopachrome in the reaction possess the potential in cosmetic area (Campo et al. mixture was measured at 490 nm using a microplate 2004). The objectives of the present study were to reader (BioTek Synergy HT, BioTek Instruments, evaluate the whitening effects of a fucoidan-rich ex- Winooski, VT, USA). tract from U. pinnatifida, a phlorotannin-rich extract from E. cava, and GAGs from sea squirt skin. To Cell culture improve the whitening effects and to reduce the cy- B16F10 mouse melanoma cells (ATCC® CRL-6475™) totoxicities of these compounds, they were mixed in were purchased from ATCC (American Type Culture different ratios and tested to select the mixture that Collection, Manassas, VA, USA) and grown in conferred optimal whitening without cytotoxicity. DMEM supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were incubated in an atmosphere of 5% Materials and methods CO at 37 °C and were sub-cultured every 4 days. Chemicals and reagents Cells for experiments were seeded at a concentration Dimethyl sulfoxide (DMSO), 3-(4-5-dimethyl-2yl)-2-5- of 5 × 10 cells/mL. diphynyltetrasolium bromide (MTT), mushroom tyrosin- ase, and alpha-melanocyte-stimulating hormone (α-MSH) were purchased from Sigma Co. (St. Louis, MO, USA). Cell viability assay Dulbecco’s modified Eagle medium (DMEM), penicillin/ Cell viability was quantified by a colorimetric MTT streptomycin, and fetal bovine serum (FBS) were pur- assay (Wang et al. 2017;Wangetal. 2018). Briefly, chased from Gibco BRL (Life Technologies, Burlington, B16F10 cells were seeded in a 96-well plate and incu- ON, Canada). Antibodies against tyrosinase, tyrosinase- bated for 24 h. The cells were treated with different related protein-1 and protein-2 (TRP-1 and TRP-2), and concentrations of test samples and incubated for 72 h. microphthalmia-associated transcription factor (MITF) MTT solution (50 μL, 2 mg/mL) was added to each were purchased from Santa Cruz Biotechnology (Santa well and incubated for 3 h. The supernatant was aspi- Cruz, CA, USA). Anti-mouse and anti-rabbit IgG were rated, 150 μL of DMSO was added to each well, and purchased from Cell Signaling Technology (Beverly, MA, the absorbance was measured at 540 nm using a mi- USA). All other chemicals were of analytical grade. croplate reader. Preparation of fucoidan-rich extract, phlorotanin-rich extract, and glycosaminoglycans Measurement of cellular melanin content U. pinnatifida was hydrolyzed with Celluclast. The B16F10 cells were seeded in a 6-well plate and incu- polysaccharide fraction was precipitated with ethanol bated for 24 h. The cells were treated with various con- and referred to as UPEF. UPEF contains 36.10 ± 3.20% centrations of test samples and stimulated with α-MSH fucoidan. An 80% ethanol extract of E. cava was pre- (50 nM). After 72 h, the cells were washed with ice-cold pared (ECE), and it contained 26.85 ± 0.16% phenols. PBS and harvested. The harvested cells were incubated The sea squirt skin was hydrolyzed with Celluclast, at 80 °C for 1 h in 1 mL of 1-N NaOH containing 10% and glycosaminoglycans (GAGs) were separated and DMSO. The absorbance of the supernatant was Wang et al. Fisheries and Aquatic Sciences (2019) 22:11 Page 3 of 8 measured at 490 nm using a microplate reader (Heo et Results al. 2010). Tyrosinase inhibition by UPEF, ECE, and GAGs The effects of UPEF, ECE, and GAGs on tyrosinase activity were examined by measuring L-tyrosine hydroxyl- Western blot analysis ation. Arbutin was used as a positive control. As Fig. 1 The effect of the test samples on the expression of shows, UPEF, ECE, and GAGs inhibited tyrosinase activity melanogenesis-related proteins including MITF, tyrosinase, in a dose-dependent manner. ECE showed the strongest TRP-1, and TRP-2 was assessed by Western blot analysis as inhibition of the three agents, and it inhibited tyrosinase described previously (Kim et al. 2013). In brief, B16F10 cells activity by 64.33% at a concentration of 100 μg/mL. were incubated with various concentrations of the test sam- ple and stimulated with α-MSH (50 nM). After 72 h, cells were harvested and lysed. The protein content of each sam- Cytotoxicity of UPEF, ECE, and GAGs on B16F10 cells ple was measured with a BCA™ kit. The proteins (50 μg) The cytotoxicities of UPEF, ECE, and GAGs on B16F10 were separated by SDS-PAGE and transferred onto nitro- cells were examined by MTT assay, and the results are cellulose membranes. The membranes were incubated in summarized in Fig. 2a. As the results show, ECE caused blocking buffer (5% skim milk) and then with primary anti- significant cytotoxicity in B16F10 cells. In addition, bodies for 16 h at 4 °C. The membranes were then incu- UPEF and GAGs were slightly toxic to B16F10 cells at bated with secondary antibody at room temperature for 3 high concentration (100 μg/mL). h. Finally, the proteins were visualized using an ECL West- ern blotting detection kit and exposure to X-ray film. Effects of UPEF, ECE, and GAGs on melanin synthesis in α- MSH-stimulated B16F10 cells Statistical analysis As Fig. 2b indicates, the melanin content of cells not All experiments were performed in triplicate. The data were stimulated with α-MSH is referred to as 100%, and expressed as means ± standard errors (S.E). The mean the melanin content of cells stimulated with α-MSH values of each experiment werecompared using one-way increased by 80%. The melanin content of cells ANOVA. Significant differences between the means were treated with UPEF, ECE, and GAGs decreased in a determined by the Duncan test. A p value < 0.05 was dose-dependent manner. These results indicated that considered to be statistically significant, and degrees of sig- all three test substances inhibited melanogenesis in α- nificance were indicated as follows: *p < 0.05, **p <0.01, MSH-stimulated B16F10 cells, and ECE showed the ### ***p < 0.001, and p <0.001. strongest effect. Fig. 1 Effect of UPEF, ECE, and GAGs on mushroom tyrosinase activity. Tyrosinase activity was measured by colorimetric assay. Dopachrome was measured at 490 nm using a microplate reader. The data are expressed as the means ± S. E (n =3) Wang et al. Fisheries and Aquatic Sciences (2019) 22:11 Page 4 of 8 Fig. 2 Cytotoxicity and melanin synthesis in UPEF-, ECE-, and GAGs-treated B16F10 cells. a Cytotoxicity on B16F10 cells; b melanin synthesis in α- MSH-stimulated B16F10 cells. The data are expressed as the means ± S. E (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 as compared to the α- ### MSH-treated group and p < 0.001 as compared to the control group Tyrosinase inhibition by combinations of UPEF, ECE, and combinations in B16F10 cells were measured by MTT GAGs assay. As Fig. 4a shows, most combinations were UPEF, ECE, and GAGs were combined in various ratios slightly toxic to B16F10 cells, and UPEF to ECE to by volume, and the effect on tyrosinase activity was mea- GAGs at 2:6:2 (UEG-262) showed the strongest cyto- sured. As Fig. 3 shows, tyrosinase activity decreased as toxicity. On the other hand, UEG-451 showed no cyto- the ECE ratio in the mixture increased. In addition, the toxicity in B16F10 cells. inhibitory activities of the test agents in combination were stronger compared to each agent alone at the same concentration. Effect of test agents in combination on melanin synthesis in α-MSH-stimulated B16F10 cells The effects of the test agents in combination on melano- Cytotoxicity of UPEF, ECE, and GAGs in combination on genesis were assessed by measuring melanin synthesis in B16F10 cells α-MSH-stimulated B16F10 cells. As the results show Using the tyrosinase inhibition conferred by the test (Fig. 4b), all combinations significantly reduced melanin agents in combination, seven combinations (UEG-262, synthesis in α-MSH-stimulated B16F10 cells, especially UEG-271, UEG-352, UEG-361, UEG-433, UEG-451, UEG-451. These results show that UPEF, ECE, and and UEG-721) were selected to test the effects on mela- GAGs in combination inhibited melanogenesis, and 4:5: nogenesis in B16F10 cells. The cytotoxicities of these 1 is the optimum ratio. Wang et al. Fisheries and Aquatic Sciences (2019) 22:11 Page 5 of 8 Fig. 3 The effects of UPEF, ECE, and GAGs in combination on mushroom tyrosinase activity. Tyrosinase activity was measured by colorimetric assay. Dopachrome was measured at 490 nm using a microplate reader. The data are expressed as the means ± S. E (n =3) Fig. 4 Cytotoxicity and melanin synthesis in B16F10 cells treated with UPEF, ECE, and GAGs in combination. a Cytotoxicity on B16F10 cells; (b) melanin synthesis in α-MSH-stimulated B16F10 cells. The data are expressed as the means ± S. E (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 as ### compared to the α-MSH-treated group and p < 0.001 as compared to the control group Wang et al. Fisheries and Aquatic Sciences (2019) 22:11 Page 6 of 8 Fig. 5 Effect of UEG-451 on tyrosinase, TRP-1, TRP-2, and MITF expression in α-MSH-stimulated B16F10 cells. a The effect of UEG-451 on tyrosinase, TRP-1, TRP-2, and MITF expressions; (b) the relative amounts of tyrosinase, TRP-1, TRP-2, and MITF. The relative amounts of tyrosinase, TRP-1, TRP-2, and MITF were normalized to β-actin. The data are expressed as the means ± S. E (n = 3). **p < 0.01 and ***p < 0.001 as compared to ### the α-MSH-treated group and p < 0.001 as compared to the control group Effect of UEG-451 on tyrosinase, TRP-1, TRP-2, and MITF are made from natural or chemosynthetic materials, are expression in α-MSH-stimulated B16F10 cells commercially available. However, some of these mate- Based on its cytotoxicity and inhibition of melanogenesis, rials possess side effects or are toxic because they con- UEG-451 was selected for further research to evaluate the tain substances such as hydroquinone and heavy metals. mechanisms of its anti-tyrosinase and anti-melanogenesis Thus, more research is devoted to searching for safe and activities. The effect of UEG-451 on the expression of effective whitening agents from natural sources. tyrosinase, TRP-1, TRP-2, and MITF was examined by Many studies have reported the effects of plant ex- Western blot analysis. As shown in Fig. 5, the expressions tracts or compounds isolated from plants on melanogen- of tyrosinase, TRP-1, TRP-2, and MITF were increased by esis (Arung et al. 2011; Chan et al. 2011). Arung et al. α-MSH stimulation and the expressions of these proteins (2011) isolated quercetin and its derivatives from Allium were reduced in cells pre-treated with different concentra- cepa and investigated their anti-melanogenesis effects tions of UEG-451. (Arung et al. 2011). Chan et al. (2011) evaluated the tyrosinase inhibition and melanin synthesis inhibition in Discussion B16F10 cells of Sargassum polycystum ethanolic extracts In Asian cultures, lighter skin tones are considered more and their fractions (Chan et al. 2011). In the present desirable. To meet the needs of the many women who study, we evaluated melanogenesis inhibition conferred by suffer from hyperpigmentation, whitening agents have a fucoidan-rich extract, a phlorotannin-rich extract, and been developed for cosmetic and medical applications. GAGs obtained from seaweed and sea squirt, and investi- Various whitening cosmetics or cosmeceuticals, which gated their whitening effects when in combination. Wang et al. Fisheries and Aquatic Sciences (2019) 22:11 Page 7 of 8 The effects of UPEF, ECE, and GAGs on commercial three agents inhibited tyrosinase and melanin synthesis mushroom tyrosinase were investigated, and the results in α-MSH-stimulated B16F10 cells and that they can act indicated that all samples possess tyrosinase inhibitory ac- synergistically when in combination. In addition, UPEF, tivity with ECE showing the strongest activity of the three ECE, and GAGs in the ratio of 4:5:1 (UEG-451) showed (Fig. 1). In addition, UPEF, ECE, and GAGs significantly the strongest activity and was not toxic. These results reduced α-MSH-induced melanin synthesis in B16F10 suggest UEG-451 may be an ideal whitening agent for cells in a dose-dependent manner (Fig. 2). ECE showed use in the medical and cosmetic industries. stronger melanin synthesis inhibition activity than UPEF Acknowledgements and GAGs, and the melanin content of cells treated with The authors wish to thank the Ministry of Trade, Industry and Energy 100 μg/mL of ECE was lower than the unstimulated cells. (MOTIE), and Korea Institute for Advancement of Technology (KIAT) for the support for this study. These results demonstrated that ECE is a potent in- hibitor of melanin synthesis in α-MSH-stimulated and Authors’ contributions non-stimulated B16F10 cells. However, all three sam- LW, JYK, and YJJ designed this study and wrote the manuscript. LW, YRC, ples were cytotoxic to B16F10 cells, especially ECE. HWY, and HGL performed the experiments and analyzed the data. LW and YRC contributed equally to this study. All authors read and approved the Therefore, we mixed the test agents in different ratios final manuscript. and compared the whitening effects and toxicities of the combinations to those of the agents alone. Funding The inhibition of tyrosinase and melanin synthesis This research is supported by “Rediscover of the Past R&D Result” through the Ministry of Trade, Industry and Energy (MOTIE), and Korea Institute for conferred by the combinations was stronger than each Advancement of Technology (KIAT) (Grant No.: N0002151, Project: The single agent at the same concentrations (Fig. 1 and Fig. 3; commercialization of anti-aging products (Aquato Return 20). Fig. 2b and Fig. 4b); UEG-451 showed the strongest ac- Availability of data and materials tivity and no toxicity, with 71.10% of control tyrosinase All data sets generated and analyzed during the current study are available activity and a 115.24% reduction in α-MSH-stimulated from the corresponding author on reasonable request. melanin synthesis at 50 μg/mL. Combining the agents at an optimal ratio may be an ideal and effective way to im- Ethics approval and consent to participate Not applicable. prove bioactivity and reduce cytotoxicity. Melanogenesis is regulated by enzymes including tyro- Consent for publication sinase, TRP-1, and TRP-2. Tyrosinase is considered to Not applicable. be the rate-limiting enzyme of melanin biosynthesis and Competing interests represents the major regulatory step in melanogenesis The authors declare that they have no competing interests. (Maeda et al. 1997). Thus, the inhibition of related en- zymes is the most common approach to developing Received: 9 April 2019 Accepted: 17 May 2019 skin-whitening agents. The present results revealed that UEG-451 inhibited tyrosinase and reduced melanin syn- References thesis in α-MSH-stimulated B16F10 cells. In addition, Ahn G-N, Kim K-N, Cha S-H, Song C-B, Lee J, Heo M-S, Yeo I-K, Lee N-H, Jee Y-H, Western blot results demonstrated that the expressions Kim J-S. Antioxidant activities of phlorotannins purified from Ecklonia cava on free radical scavenging using ESR and H2O2-mediated DNA damage. Eur of tyrosinase, TRP-1, and TRP-2 in UEG-451-treated Food Res Technol. 2007;226:71–9. cells decreased in a dose-dependent manner compared Arung ET, Furuta S, Ishikawa H, Kusuma IW, Shimizu K, Kondo R. Anti- to non-treated cells (Fig. 5a and b). The tyrosinase fam- melanogenesis properties of quercetin-and its derivative-rich extract from Allium cepa. 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Fisheries and Aquatic SciencesSpringer Journals

Published: May 31, 2019

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